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Differential Notch signalling distinguishes neural stem cells from intermediate progenitors

Abstract

During brain development, neurons and glia are generated from a germinal zone containing both neural stem cells (NSCs) and more limited intermediate neural progenitors (INPs)1,2,3. The signalling events that distinguish between these two proliferative neural cell types remain poorly understood. The Notch signalling pathway is known to maintain NSC character and to inhibit neurogenesis, although little is known about the role of Notch signalling in INPs. Here we show that both NSCs and INPs respond to Notch receptor activation, but that NSCs signal through the canonical Notch effector C-promoter binding factor 1 (CBF1), whereas INPs have attenuated CBF1 signalling. Furthermore, whereas knockdown of CBF1 promotes the conversion of NSCs to INPs, activation of CBF1 is insufficient to convert INPs back to NSCs. Using both transgenic and transient in vivo reporter assays we show that NSCs and INPs coexist in the telencephalic ventricular zone and that they can be prospectively separated on the basis of CBF1 activity. Furthermore, using in vivo transplantation we show that whereas NSCs generate neurons, astrocytes and oligodendrocytes at similar frequencies, INPs are predominantly neurogenic. Together with previous work on haematopoietic stem cells4, this study suggests that the use or blockade of the CBF1 cascade downstream of Notch is a general feature distinguishing stem cells from more limited progenitors in a variety of tissues.

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Figure 1: CBF1 signalling heterogeneity in the telencephalic VZ.
Figure 2: Analysis of CBF1 function in NSCs and INPs.
Figure 3: In vitro analysis of EGFP hi and EGFP lo/neg cells.
Figure 4: Differentiation analysis of EGFP hi and EGFP lo/neg cells.

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Acknowledgements

We thank D. Hayward, D. Johns, E. Manet, T. Haydar, A. Ayoub and T. Ohtsuka for plasmids; J. Corbin for the PLAP mice; L. Blosser and A. Tam for cell sorting; R.-J. Zhao and J. Kim for technical assistance; and R. Wechsler-Reya, T. Reya, M. Starz-Gaiano, T. Haydar and S. Temple for discussions. K.M. was supported by a fellowship from the Japan Society for the Promotion of Science. This work was supported by grants from the Burroughs Wellcome Fund, the Sidney Kimmel Foundation for Cancer Research, and the National Institute of Neurological Disorders and Stroke (all to N.G.).

Author Contributions K.M. performed in utero electroporations, adherent and neurosphere cell culture experiments, quantitative RT–PCR analysis, γ-secretase inhibition and shRNA experiments. K.Y. generated and validated the TNR line, established flow cytometry staining protocols, performed γ-secretase inhibition and MEF experiments, and did the Nestin tissue staining. L.D. characterized the in vivo expression pattern of EGFP in the telencephalon of TNR embryos, performed in vivo cell transplantations, and did the CD133 tissue staining. A.T. performed clN1, EGFP and CD133 immunostainings. N.G. conceived of the TNR line, oversaw the project, and prepared the manuscript.

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Correspondence to Nicholas Gaiano.

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N.G. and K.Y. are listed by the Johns Hopkins Technology Transfer office as the inventors of the TNR mouse line; licensing of that line to for-profit entities could result in remuneration for the inventors.

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Mizutani, Ki., Yoon, K., Dang, L. et al. Differential Notch signalling distinguishes neural stem cells from intermediate progenitors. Nature 449, 351–355 (2007). https://doi.org/10.1038/nature06090

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